Prokaryotic Expression,Purification And Identification Of Hadal Snailfish Hsp90

Heat shock protein 90(Hsp90)is a kind of molecular chaperone protein with a monomer molecular weight of about 90 kD,which is widely present in prokaryotes and eukaryotes.This kind of protein is highly conserved and abundant in cells.They are necessary for cell survival.They are involved in the folding and regulation of various proteins related to signal transduction and cell cycle regulation,and play an important role in maintaining protein homeostasis.Because its client proteins are mostly related to cell proliferation and tumor progression,Hsp90 has become a key target for anti-tumor drug design.Hsp90 has two subtypes,? and ?,in eukaryotic cells,and is active as a dimer of?-? or ?-?.The N-terminus is an ATP-binding domain with ATPase activity and can be combined with inhibitors or co-molecule chaperones;the intermediate domain is mainly combined with customer proteins and co-molecule chaperones;the C-terminal domain is mainly responsible for its dimerization.The analysis of the existing electron microscope and crystal structure shows that the function of Hsp90 is completed by the N-terminal combination of ATP-driven transient dimerization and the dimerization opened after the ATP hydrolysis.This conformational change from closed to open state is completed.There are five copies of the hsp90 gene in the genome of a Hadal Snailfish(Pseudoliparis swirei)living in the Mariana Trench below 7000 m above sea level,and four of them occur at a conserved site in the N-terminal domain repeated fixed mutations.According to the analysis of homology modeling results,the mutation site is close to the ATP binding pocket and may interact with some of the amino acids that make up the active pocket,thereby affecting Hsp90 activity.It is speculated that the mutation may cause structural and functional changes to adapt to the deep seahydrostatic pressure of this species of Snailfish.In order to reveal the effect of this unique mutation on the structure and function of Hsp90 of Pseudoliparis swirei,it is necessary to obtain the Hsp90 protein of this species first.Because the experimental raw materials are difficult to obtain,this paper attempts to express and purify the protein by means of exogenous recombinant expression.At present,when the structure or function of Hsp90 protein is studied,the most common one is to use the Escherichia.coli(E.coli)expression system for exogenous expression to obtain the protein,so we chose to use the pET-28 a plasmid to construct a recombinant vector,and induced expression in BL21(DE3).To facilitate protein separation and purification,His and Strep tags were added to the N-terminu of Hsp90 protein.Western Blot and mass spectrometry identification analysis verified that the target protein was expressed in E.coli.Through a variety of separation and purification methods such as affinity chromatography,anion exchange chromatography as well as gel exclusion chromatography,we obtained Hsp90 protein with a certain purity,and initially observed the protein structure using transmission electron microscopy.Natural Hsp90 has ATPase activity,and we measured the activity of Hsp90 hydrolyzing ATP through a sensitive enzyme-coupled assay system.The experimental results show that the Hsp90 protein expressed in prokaryotic cells has enzyme activity.This thesis laid an experimental foundation for exogenous expression and purification of Hsp90 protein of this species,and provided an experimental basis for further optimization of the experimental process to improve protein purity for structural biology research.